The concentration of lipoproteins in blood is important in clinical tests. Lipoproteins can be divided into two groups—high density lipoproteins (HDL) and low density lipoproteins (LDL), each of which having different biological functions. As a measure of the lipoprotein content in blood, cholesterol associated with the lipoproteins is measured. In biological samples like blood, cholesterol is present in the lipoproteins in the form of cholesterol esters.
To measure the lipoprotein-associated cholesterol levels, the cholesterol esters are split by enzymes such as cholesterol esterase. Once freed, cholesterol then is determined. The cholesterol concentration in blood may be measured using an enzyme having specificity to cholesterol such as, for example, cholesterol oxidase (ChOx).
ChOx has been isolated from various organisms, and it has been suggested that cholesterol may be analyzed using such enzymes. ChOx is a flavin adenine dinucleotide (FAD)-dependent enzyme that catalyzes a reaction where cholesterol is oxidized to generate cholest-4-en-3-one, thereby generating the reduced form of FAD, FADH2. FADH2, in turn, transmits electrons to an electron acceptor and is converted to its oxidized form. In the presence of oxygen, FADH2 preferentially transmits electrons to oxygen molecules rather than to artificial electron acceptors (also referred to as mediators or electron mediators). Thus, when cholesterol is assayed by ChOx with mediators, the assay results will be greatly affected by the dissolved oxygen level in the reaction system. Such a disadvantage will be particularly noted in clinical tests of blood samples by a point-of-care testing device utilizing an artificial electron acceptor. Therefore, enzymes used for enzyme biosensor test strips employing artificial electron mediators desirably have low activity toward oxygen.
For the foregoing reason, there is a need for an enzyme, in particular, a ChOx having an activity that is less affected by the dissolved oxygen level.